Human Interleukin-3(Il-3) variants and their use to promote or antagonize IL-3-mediated processes

ABSTRACT

This invention relates to human IL-3 variants. The human IL-3 variants are used in pharmaceutical compositions, in methods for modulating proliferation, differentiation or functional activation of cells expressing the IL-3 receptor, and in methods of treatment.

This invention relates to variants and mutants of human interleukin-3(hIL-3), and in particular it relates to hIL-3 va sequence of wild-typehIL-3 is varied in order to obtain useful changes in activity,particularly in binding to IL-3 receptors and in biological function.

Human IL-3 is a T-cell-derived glycoprotein of Mr 23-30 kd whichpromotes the proliferation and differentiation of haemopoieticprogenitor cells, including megakaryocytes, eosinophils, basophils,neutrophils, monocytes and T-lymphocytes. It also induces the growth andthe functional activation of more mature cells, including eosinophils,basophils and monocytes. The cDNA of hIL-3 has been cloned, and themature protein of 133 amino acids has been produced in recombinant form.The human IL-3 receptor comprises at least two components, an a chainwhich binds IL-3 with low affinity only, and a β chain which allows highaffinity binding when co-expressed with the α chain (Kitamura T, Sato N,Arak K-I and Miyajima A, 1991, Cell 66, 1165-1174).

Subsequent structure-activity relationship studies of hIL-3 have beenperformed by functional analysis of hIL-3 deletion and substitutionvariants (Lokker et al, 1991a, J. Biol. Chem. 266, 10624-10631; 1991b,EMBO J. 10, 2125-2131) using recombinant hIL-3 variants generated bysite-directed mutagenesis and expression in Escherichia coli. In thiswork, the variants were analysed for their ability to bind to the IL-3receptor and to induce the proliferation of the human IL-3 -dependentcell line M-07. These studies initially showed that hIL-3 residues Pro33 and Leu 34 are essential for modulating the biological activity ofhIL-3, and that certain substitution variants at residues 33 and 34,particularly the variant in which Pro 33 was substituted with Gly (Gly33), showed an enhanced proliferation activity without a significantmodification in its receptor binding capacity (Lokker et al, 1991asupra). Subsequent studies which extended the structure-activity-relationship studies showed that the hIL-3 residue Leu 111, and possiblyalso Lys 110, form part of an active site. Thus, substitution of Lys 110with either Glu or Ala resulted in variants with substantially reducedactivity in receptor binding and proliferation assays. Similarly,variants where Leu 111 was substituted by Pro or Met were totallyinactive in these assays (Lokker et al, 1991b supra).

It has now been discovered that variants or mutants of hIL-3 in whichone or more amino acids in or adjacent to the predicted "D" or fourthpredicted α-helix of hIL-3 is/are replaced with another amino acid showenhanced biological activity when compared with wild-type hIL-3. Thisenhanced biological activity is paralleled by enhanced binding to thespecific a chain of the IL-3 receptor, and suggests that the variants ormutants may be used as therapeutic agents.

According to a first aspect of the present invention, there is provideda human IL-3 variant or mutant, characterised in that one or more aminoacids in or adjacent to the predicted "D" or fourth predicted α-helix ofhIL-3 is/are replaced by another amino acid.

In one embodiment of this aspect of the invention, there is provided ahuman IL-3 variant or mutant, characterised in that amino acid 101 (Asp)and/or amino acid 116 (Lys) is/are replaced by another amino acid.

Particularly preferred variants or mutants in accordance with thisaspect of the invention are:

hIL-3 (Ala¹⁰¹)

hIL-3 (Val¹¹⁶)

hIL-3 (Ala¹⁰¹, Val¹¹⁶)

In addition, it has also been found that replacement of one or moreamino acids in the predicted "A" or first predicted α-helix with anotheramino acid, particularly replacement of amino acids 21, 22 and 25,results in loss of IL-3 activity to high affinity IL-3 receptorsindicating that these residues form part of another IL-3 active part. Ithas, however, been shown that these biologically inactive mutants stillretain binding ability to the α chain of the IL-3 receptor. The loss ofbiological activity suggests that these mutants may be used asantagonists.

According to a second aspect of this invention, there is provided ahuman IL-3 variant or mutant, characterised in that one or more aminoacids in the predicted "A" or first predicted α-helix of hIL-3 is/arereplaced by another amino acid.

In one embodiment of this aspect of the invention, there is provided ahuman IL-3 variant or mutant, characterised in that amino acid 21 (Asp),amino acid 22 (Glu) and/or amino acid 25 (Thr) is/are replaced byanother amino acid.

Particularly preferred variants or mutants in accordance with thisaspect of the invention are:

hIL-3 (Ala²¹, Leu²², Ala²⁵)

hIL-3 (Ala²¹, Leu²²)

hIL-3 (Ala²¹)

hIL-3 (Arg²¹)

hIL-3 (Leu²²)

hIL-3 (Arg²²)

hIL-3 (Ala²⁵)

In yet another aspect, this invention provides a human IL-3 variant ormutant which is characterised in that it combines the two sets ofvariations or mutations broadly described above, that is amino acidreplacement is effected in both the "A" α-helix and in or adjacent tothe "D" α-helix. These variants or mutants will combine the antagonistactivity resulting from loss of biological activity with increasedaffinity, resulting in enhanced IL-3 antagonist potency.

Particularly preferred variants or mutants in accordance with thisaspect of the invention are:

hIL-3 (Ala¹⁰¹, Val¹¹⁶, Arg²²)

hIL-3 (Ala¹⁰¹, Val¹¹⁶, Ala²¹, Leu²²)

The present invention also extends to the use of the mutants or variantsas described above as therapeutic agents. Thus, these mutants orvariants may be provided as active components in therapeuticcompositions, together with one or more pharmaceutically acceptablecarriers or diluents.

The therapeutic use of the variants or mutants of this invention mayinclude, for example, modulation of proliferation and differentiation ofhaemopoietic progenitor cells or of growth and functional activation ofmature haemopoietic cells. This modulation may be as an agonist or anantagonist of IL-3 function. In its broadest sense, the therapeutic useof these variants or mutants extends to modulation of the function ofall cells that express or are made to express IL-3 receptor, includingboth haemopoietic cells and non-haemopoietic cells such as non-myeloidcells expressing or made to express IL-3 receptor.

Further details of the present invention are set out in the followingExample, and in the accompanying Figures.

IN THE FIGURES:

FIG. 1 shows the predicted structure of hIL-3, including the fourpredicted α-helix structures labelled A, B, C and D. Predicted positionsof amino acid residues 101 and 116 are identified.

FIG. 2 shows the proliferation of chronic myeloid leukaemic (CML) cells,as measured by [³ H] thymidine incorporation, in the presence ofdifferent concentrations of IL-3 analogs.

FIG. 3 shows the stimulation of monocyte adherence by differentconcentrations of IL-3 mutants.

FIG. 4 shows the stimulation of histamine release by different IL-3mutants.

FIG. 5 shows the ability of IL-3 mutants to compete for ¹²⁵ I-IL-3binding to the high affinity receptor of monocytes. The deriveddissociation constants (K_(d)) for each mutant is also shown.

FIG. 6 shows the ability of E. coliIL-3 mutants to compete for ¹²⁵I-IL-3 binding to the cloned IL-3R α chain expressed in COS celltransfectants. Note that IL-3 (Ala¹⁰¹, Val¹¹⁶) is more potent atcompeting for binding than wild-type IL-3.

FIG. 7 shows competition of E. coliIL-3 mutants for ¹²⁵ I-IL-3 bindingto the reconstituted IL-3 high affinity receptor. In this case both thecloned α and α chains were expressed in COS cells.

FIG. 8 shows the proliferation of chronic myeloid leukaemic cells bydifferent IL-3 analogs. Note that replacement of glutamic acid inposition 22 by leucine or arginine results in an inactive molecule.

EXAMPLE Materials and Methods

1. Site Directed Mutagenesis of Human IL-3

Human IL-3 mutants were constructed using either site-directedmutagenesis or the polymerase chain reaction PCR.

Substitution of amino acid residue 101 (aspartic acid) by alanine andamino acid 116 (lysine) by valine was performed by oligonucleotidesite-directed mutagenesis. The method used was that of Zoller and Smith(1984, DNA, 3, 479).

The oligonucleotide sequences used were:

(a) Asp(101)-Ala: ##STR1## (b) Lys(116)-Val: ##STR2##

(note: altered residue(s) double underlined)

Site-directed mutagenesis involved annealing a mutagenic oligonucleotideto a single stranded M13 vector containing a hIL-3 cDNA constructedsynthetically (Phillips el. al., 1989, Gene, 84, 501-507). Addition ofdNTPs and DNA polymerase (Klenow fragment) allowed extension from themutant primer along the M13 template. A primer specific for the M13sequence (USP) was added to increase efficiency of the reaction. Theresulting heteroduplex was transformed into an E.coli strain, JM101.Resulting plaques were lifted onto nitrocellulose filters and screenedwith the 32p-labelled mutagenic oligonucleotide. Single stranded DNA wasprepared from positive plaques and sequenced to confirm the mutation(Zoller and Smith, supra).

A two part polymerase chain reaction was used to create mutants in thedouble stranded IL-3 construct, pJLA⁺ IL-3 (Phillips et. al., supra).Three primers were involved. Two lay outside of the IL-3 gene and thethird was the mutagenic oligonucleotide. In the first step the outsideprimer that binds to the antisense strand was used with the mutagenicoligonucleotide (binds to the sense strand). Twenty five cycles of PCRwith these primers resulted in amplification of a portion of the gene.This portion contained the mutant sequence and was used as a primertogether with the other outside primer (binds to the sense strand) forthe second PCR reaction.

After construction of the mutants by site-directed mutagenesis or PCR,the double stranded DNA was digested with BamHI and SacI and cloned withan SacI/EcoRI cut DNA fragment containing SV40 polyadenylation signalsinto BamHI/EcoRI pJL4 (Gough et. al., 1985, EMBO J., 4, 645). PlasmidDNA was sequenced to confirm the presence of the IL-3 mutant sequence.

2. Transfection of IL-3 and Its Analogs.

Transient transfections were carried out in COS cells. COS cells weregrown to 50-70% confluence in Dulbecco's Modified Eagle's medium (DMEM)containing 20 mM Hepes, Penicillin, Gentomycin and supplemented with 10%fetal calf serum (FCS). Cells were harvested with trypsin/EDTA,centrifuged and immediately before use resuspended in 20 mMHEPES-buffered saline containing 6 mM glucose to 1×10⁷ cells/ml.

DNA constructs were introduced into COS cells by electroporation (Chuet. al., 1987, Nucleic Acids Res., 15, 1311-1376). For eachtransfection, 20 μg of pJLA⁺ IL-3 plasmid DNA, 25 μg sonicated salmonsperm DNA and 50 μl FCS were mixed with 5×10⁶ COS cells. The mixture waselectroporated using a Bio-Rad Gene Pulser before being plated out inDMEM + 10% FCS. After a 24 hour incubation period the medium wasreplaced with FCS-free DMEM and incubated for a further 72 hours beforethe conditioned medium was harvested and assayed for IL-3 protein.

3. Visualisation of IL-3 Protein.

COS cell supernatants containing IL-3 were size-fractionated bySDS-12.5% PAGE and then protein transferred to nitrocellulose. IL-3protein detection was carried out by Western Blot analysis usinganti-human IL-3 antibodies and visualized by autoradiography after theaddition of ¹²⁵ I-protein A.

4. Quantitation of IL-3 Protein.

The mount of IL-3 protein present in COS supernatants was quantitated bya radioimmunoassay (RIA). A competitive RIA was developed using ¹²⁵-I-labelled IL-3 and a polyclonal anti-IL-3 serum (gift from Dr S Clark,Genetics Institute). IL-3 was labelled with ¹²⁵ I by the iodinemonochloride method as described (Contreras et. al., 1983, Meth.Enzymol. 92, 277-292). COS cell supernatants (50 μl) were incubated withrabbit anti-IL-3 serum (50 μl of 1:10,000 dilution) in Eppendorfmicrotubes. After 4 hr incubation at 4° C., 0.1 ng of ¹²⁵ I-IL-3 wasadded for a further 16 hr before adding 100 μl of reconstitutedanti-rabbit Immunobead reagent (Bio-Rad Laboratories, Richmond, Calif.)for 4 hr. The mixtures were then washed twice with PBS, and the pelletwas resuspended in 200 μl of PBS and transferred to 3DT tubes forcounting in a gamma-counter (Packard Instrument Company, Meriden,Conn.). The amount of IL-3 protein was calculated from a standard curveconstructed with known amounts of IL-3.

Wild type IL-3 and IL-3 (Ala¹⁰¹, Val¹¹⁶) protein produced in E. coliwere also quantitated directly by scanning densitometry (Fazekas de St.Groth et at., 1963, Biochim. Biophys. Acta., 71,377-391). Briefly,proteins were electrophoresed on 15% SDS PAGE and stained with Coomassiebrilliant blue R250. Samples of wild type IL-3, IL-3 (Ala¹⁰¹, Val¹¹⁶) orRNAse standards were electrophoresed over a concentration range of 0.5-5μg in duplicate and the gel then analysed using an LKB-PharmaciaUltrascan XL scanning laser densitometer. Data analysis was performedwith GSXL densitometer software. The protein concentrations of theunknown samples were calculated using the area under the peak, relativeto known amounts of RNase standards using the same absorbancecoefficient. In some cases direct protein quantitation was alsoperformed by HPLC peak integration by calculating the area under theIL-3 peak using the extinction coefficient of 0.83 AU.ml/mg. The valuesobtained with each method were very similar. An IL-3 preparation (giftfrom Genetics Institute) at 0.6 μg/ml (by amino acid analysis) measured0.59±0.1 (mean ± SD) lag/ml by scanning laser densitometry, and 0.6±0.07μg/ml by radioimmunoassay. In parallel, an IL-3 (Ala¹⁰¹, Val¹¹⁶)concentration of 1.45±0.06 μg/ml by scanning laser densitometry comparedwith 1.32±0.2 μg/ml by HPLC peak integration, and 1.35±0.2 μg/ml by RIA.

5. Stimulation of Hemopoietic Cell Proliferation.

Two types of assay were performed:

(a) Colony assay: this assay measured the clonal proliferation anddifferentiation of bone marrow progenitor cells in semi-solid agar andwas carried out as described (Lopez et. al., 1985, Blood 72, 1797-1804).Briefly, low density, macrophage-depleted bone marrow cells werecultured at a concentration of 0.5 to 1×10⁵ /mL in Iscove's modifiedDulbecco's medium (IMDM, GIBCI, Grant Island, N.Y.), containing 0.33%agar (Difco, Detroit), 25% FCS (Commonwealth Serum Laboratories,Parkville, Victoria, Australia), and 20 μmol/LK2-mercaptoethanol.Different dilutions of IL-3- containing COS cell supernatants were addedto each plate. Plates were prepared in triplicate and scored afterincubation at 37° C. in 5% CO2 in a humid atmosphere for 14 days. Clonescontaining 40 cells were scored as colonies.

(b ) Proliferation of chronic myeloid leukaemic (CML) cells: PrimarilyCML cells from one patient were selected for their ability toincorporate [³ H] thymidine in response to IL-3 as described (Lopez el.al., supra ). Briefly, CML cells were placed at 2×10⁵ cells/mL freshmedium containing different concentrations of IL-3. Cells were incubatedfor 24 hours in a flat bottom 96-well NUNCLON plates (2×10⁴ cells/well)before being pulsed with [³ H] thymidine (0.5 μCi/well) for four morehours at 37° C. The cells were then harvested onto glass filters with aTitertek automated cell harvester and counted into a Beckman liquidscintillation counter. Data are expressed in cpm, and each point is themean of six replicates.

6. Stimulation of Human Monocyte Function:

(a) Monocyte purification. Monocytes were purified from the peripheralblood of normal donors, obtained from the Adelaide Red Cross TransfusionService, as previously described (Elliott et. al., 1990, J. Immunol.,145, 167-171). In brief, mononuclear cells were prepared bycentrifugation of whole blood on lymphoprep cushions (Nyegaard, Oslo,Norway) and washed twice in HBSS, 0.02% EDTA, 0.1% heat inactivated FCS(Flow Laboratories, North Ryde, Australia) and monocytes were purifiedin a Beckman J-6M/E elutriator using the Sanderson chamber, a flow rateof 12 ml/min and a constant rotor speed of 2050 rpm. Cells remaining inthe chamber after 30 min were collected, washed twice in HBSS, and usedimmediately. Using these methods, monocyte purity as assessed bymorphology and nonspecific-esterase staining was always >90% andusually >95%. The major contaminating cell types were lymphocytes andgranulocytes (principally basophils).

(b) Adhesion assay. Adhesion was measured by an isotopic methodessentially as described (Elliott et. al., supra). In brief, purifiedmonocytes (0.5 to 1×10⁸) were resuspended in 1 ml RPMI 1640 with 0.1%FCS and antibiotics and incubated for 30 min at 37° C. with 500 μCi⁵¹ Crin the form of sodium chromate (Amersham International.,Buckinghamshire, England). Cells were washed thrice in RPMI 1640 andresuspended in culture medium consisting of RPMI 1640, 10% FCS,antibiotics, and 0.2% sodium bicarbonate. For measurement of adhesion, 1to 2.5×10⁵ monocytes were aliquotted per well in 96-well microtitreplates (Nunc, Karnstrup, Denmark) together with stimuli or controlmedium to a total volume of μl, and incubated for the indicated periods.Monocyte settling under these conditions were observed to be completewithin 10 min of incubation. At harvest, samples of supernatant weretaken to assess spontaneous ⁵¹ Cr release (usually <10% ofcell-associated radioactivity), wells were washed three times with RPMI1640 at 37° C., and residual adherent cells lysed in 10 mMTris-hydrochloride, and 1% Nonident p40 detergent (Sigma). Lysates weretransferred to tubes and counted in a Packard auto-gamma 5650. Percentadherence was calculated according to the formula: ##EQU1##

7. Histamine Release Assay.

This was carried out as previously described (Lopez et. al., 1990, J.Cell. Physiol., 145, 69-77). Briefly, basophils were obtained from theperipheral blood of normal individuals after dextran sedimentation andcentrifugation over Lymphoprep. The percentage of basophils in thesepreparations varied between 0.2% and 10%. In 300 μl 2×10⁴ cells wereincubated with 2 μg/ml of purified human IgE. IgE-sensitised cells weremixed with a goat IgG antihuman IgE (Cappel 0101-0061) and rhIL-3, in afinal volume of 500 μl. After incubation for 60 min at 37° C., the cellswere centrifuged and 350 μl aliquots removed and stored at -20° C.before assaying for histamine content. Histamine was assayed using aradioertzymatic method essentially as described (Shaff and Beavan, 1979,Anal. Biochem., 94, 425-430). Briefly, samples of 30 μl were dilutedwith an equal volume of water and mixed with a 30 μl solution comprising27.5 μl 0.1 M sodium phosphate, pH 7.9, 1.5 μl rat kidneyhistamine-N-methyltransferase, and 1.0 μl (0.5 μCi)³H-methyl-S-adensoyl-L-methionine (Dupont Net 155, low SA).Tritium-labelled methyl-histamine was extracted into chloroform/ether,dried, and counted by scintillation spectrophotometry. The cells areexpressed as nanograms of histamine per milliliter by extrapolation to astandard curve constructed with 10, 5 and 1 ng/ml of histamine (SIGMA).

8. Radioreceptor Assay:

(a) Radioiodination of hIL-3: rh IL-3 (gift from Dr. L. Park, ImmunexCorporation, Seattle, Wash.) was radioiodinated by the ICI method aspreviously described (Contreras et. al., supra). Iodinated protein wasseparated from free ¹²⁵ I by chromatography on a Sephadex G-25 PD 10column (Pharmacia, Uppsala, Sweden) equilibrated in phosphate-bufferedsaline (PBS) containing 0.02% Tween 20, and stored at 4° C. for up to 4weeks. Before use, the iodinated protein was purified from Tween andnonprotein-associated radioactivity by cation exchange chromatography ona 0.3-ml CM-Sepharose CL-6B column (Pharmacia) and stored at 4° C. forup to 5 days. The radiolabelled IL-3 retained >90% biological activityas judged from titration curves using noniondinated rh IL-3 as controls.

(b) Competition Binding assay. Freshly purified monocytes were suspendedin binding medium consisting of RPMI 1640 supplemented with 20mmol/L/HEPES and 0.5% bovine serum albumin (BSA). Typically, equalvolumes (50 μl) of 4×10⁶ monocytes, 70 pM iodinated IL-3, and differentconcentrations of IL-3 and IL-3 analog were mixed in siliconised glasstubes for 16 hr at 4° C. Cell suspensions were then overlaid on 0.2 mLFCS at 4° C. and centrifuged for 30 seconds at a maximum speed in aBeckman Microfugre 12. The tip of each tube was cut off above thevisible cell pellet and counted in a Packard Auto-Gamma 5650 (Downer'sGrove, Ill). The results are expressed as Percent competition where 100%is the competition observed in the presence of 100 fold excess nativeIL-3.

9. Competitive Displacement Assay:

Human peripheral blood monocytes were used in an assay to determine theability of mutant M37 to compete for IL-3 binding sites with wild-typeIL-3. These experiments show that M37 has 10-15 fold higher affinity forthe high affinity receptor on these cells than wild-type IL-3. This isreflected in the calculated dissociation constants:

WT: K_(d) =9.4×10⁻¹²

M37: K_(d) =5.8×10⁻¹³

10. High Affinity Binding to Cloned IL-3R α and α Chains

PolyA+ RNA was isolated from the human cord blood cell line KMT2. OligodT primed double stranded cDNA was synthesised and used as template forPCR amplification. The PCR primers were designed to amplify the completecoding region of the IL-3R alpha chain and also to amplify the codingregion of the IL-3R α chain. The PCR products were cloned into thevector pGEM-2 for sequence verification, and then into the eukaryoticexpression vector, pCDM8. The IL-3R α chain-containing plasmid wastransfected into COS cells by electroporation, either on its own or inconjunction with the IL-3R α chain-containing plasmid, and after twodays the cells were used for binding studies.

The binding of IL-3 (Ala¹⁰¹, Val¹¹⁶) produced in E. coli was compared tothat of wild type IL-3 produced in E. coli and yeast in a competitionassay using I¹²⁵ -labelled IL-3. IL-3 (Ala¹⁰¹, Val¹¹⁶) was found to have10 fold higher affinity for COS cells transfected with the IL-3R α chaincDNA and 15-fold higher affinity for COS cells transfected with theIL-3R α chain and β chain cDNAs.

RESULTS

Mutations in the C-terminus of human IL-3 resulted in the production ofthree analogs: IL-3 (Ala¹⁰¹) (referred to as M6); IL-3 (Val¹¹⁶)(referred to as M9); and IL-3 (Ala¹⁰¹, Val¹¹⁶) (referred to as M37),with increased functional activity and binding (summarised in Table).The IL-3 mutant IL-3 (Ala¹⁰¹, Val¹¹⁶) showed the greatest increase inbiological activity (15-20 fold) which correlated with increased bindingaffinity (16 fold). The likely location of the critical positions (101and 116) are indicated in the predicted four alpha helical structure ofIL-3 (FIG. 1 ) with residue 101 in a loop immediately before thepredicted fourth alpha helix, and residue 116 within the predictedfourth alpha helix.

The increased biological activity of mutants M6, M9 and M37 isdemonstrated by the stimulation of CML cells (FIG.2) and of monocyteadherence (FIG.3) where these mutants were more potent than the wildtype IL-3. An increase in the number of day 14 colonies as well asincreased histamine release from basophils (FIG.4) was also observed formutants M9 and M37. The increased ability to stimulate monocyteadherence correlated with their ability to bind to the IL-3 highaffinity receptor of monocytes (FIG.5) where M37 bound with a K_(d) of0.58 pM compared to M9 (1.5 pM), M6 (3.1 pM) and wild type IL-3 9.4 pM).The increased binding affinity was analysed on COS cells bearing thetransfected IL-3 receptor α chain or both the α and β chains. As shownin FIG. 6, M37 competed for binding more efficiently to the cellsexpressing only the α chain, thereby demonstrating that mutation in thispart of the IL-3 molecule results not only in increased potency but alsoin increased binding to a defined chain of the IL-3 receptor. FIG. 7shows that M37 has higher affinity to cloned α and β chains that arecotransfected, and IL-3 (Arg²²) (referred to as M47) has less binding tothe high affinity receptor obtained by cotransfecting the two chains.

In contrast the IL-3 mutant, IL-3 (Ala²¹, Leu²², Ala²⁵) (referred to asM25) showed lack of stimulation of CML proliferation (FIG.2) and ofmonocyte adherence (FIG.3). M25 was also negative at binding highaffinity IL-3 receptors at the concentrations tested (FIG.5). Theseresults show that M6, M9 and M37 enhance IL-3 binding as well asfunction, and that substitution of residues 21, 22 and 25 result in lossof agonistic function and high affinity binding. The contribution of thevarious mutations of M25 was analysed and the results shown in FIG. 8.It is evident that mutations at position 22 have the greatest influenceon the loss of function of this mutant and mutation of glutamic acid atposition 22 to arginine appears sufficient at abolishing IL-3 activity.This mutation, one that is likely to inhibit interaction with the αchain of the receptor (Lopez et al., 1992, EMBO J, 11:909-916), is agood potential basis of antagonists for IL-3 function. Furthermorecombinations of M37 mutations with mutations in position 22 are likelyto result in antagonists of increased affinity and therefore greaterantagonist potency. Thus, the present invention includes this model ofantagonist whereby two sets of mutations are introduced; one tofunctionally inactivate the molecule (e.g. position 22) and the other toincrease its binding to one of the receptor chains (e.g. M37 mutant).

                  TABLE                                                           ______________________________________                                        Relative biological activity and binding affinity of IL-3 mutants                                     BINDING                                               C-TERMINAL                                                                              PROLIFERATION  MONO-    Kd value                                    MUTANTS   COLONIES   CML     CYTE   (pM) +                                    ______________________________________                                        IL-3 (Ala.sup.101)                                                                      104 ± 35*                                                                             205 ±                                                                              153 ± 48                                                                          2.7                                                            82                                                       IL-3 (Val.sup.116)                                                                       420        328 ±                                                                              312   1.8                                                            116                                                      IL-3 (Ala.sup.101,                                                                      1700       1694 ±                                                                             2100   0.58                                      Val.sup.116)         281                                                      ______________________________________                                         *Mean ± SD of several experiments where a full titration was carried       out and the concentration of IL3-mutants giving 50% of biological activit     compared to that of wild type IL3.                                            + K.sub.d of wild type IL3 = 9.4 pM.                                     

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      ATATCAAGGCCGGTGACTG19                                                         (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TATAGTTCCGGCCACTGAC19                                                         (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CAGTCACCGGCCTTGATAT19                                                         (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      TTCTATCTGGTGACCCTTGAG21                                                       (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AAGATAGACCACTGGGAACTC21                                                       (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      CTCAAGGGTCACCAGATAGAA21                                                       __________________________________________________________________________

We claim:
 1. A human IL-3 (hIL-3) variant having increased affinity,relative to native hIL-3, for the high-affinity IL-3 receptor, whereinthe variations from native hIL-3 comprise the replacement of one or bothof the residues Asp¹⁰¹ and Lys¹¹⁶ by other amino acids.
 2. An hIL-3variant according to claim 1 which retains the growth-,differentiation-, or functional activation-promoting activity of nativehIL-3.
 3. An hIL-3 variant according to claim 2 which is [Ala¹⁰¹ ]hIL-3.
 4. An hIL-3 variant according to claim 2 which is [Val¹¹⁶ ]hIL-3.
 5. An hIL-3 variant according to claim 2 which is [Ala¹⁰¹, Val¹¹⁶] hIL-3.
 6. An hIL-3 variant according to claim 1 which is an antagonistof the growth-, differentiation-, or functional activation-promotingactivity of native IL-3.
 7. An hIL-3 variant according to claim 6,further comprising the replacement of one, two, or three of the residuesAsp²¹, Glu²², and Thr²⁵ in native hIL-3 by other amino acids.
 8. AnhIL-3 variant according to claim 7 which is [Ala²¹,Leu²²,Ala¹⁰¹,Val¹¹⁶ ]hIL-3.
 9. A pharmaceutical composition comprising the hIL-3 variant ofclaim 1 and a pharmaceutically acceptable carrier or diluent.
 10. Apharmaceutical composition comprising the hIL-3 variant of claim 2 and apharmaceutically acceptable carrier or diluent.
 11. A pharmaceuticalcomposition comprising the hIL-3 variant of claim 6 and apharmaceutically acceptable carrier or diluent.
 12. A method to promotethe proliferation of cells expressing a receptor for IL-3, comprisingthe step of contacting said cells with an effective amount of an hIL-3variant according to claim
 2. 13. A method to promote thedifferentiation of cells expressing a receptor for IL-3, comprising thestep of contacting said cells with an effective amount of an hIL-3variant according to claim
 2. 14. A method to promote the functionalactivation of cells expressing a receptor for IL-3, comprising the stepof contacting said cells with an effective amount of an hIL-3 variantaccording to claim
 2. 15. A method according to any one of claims 12,13, and 14, wherein said cells are present in a patient, and the methodcomprises the step of administering said variant to said patient.
 16. Amethod to antagonize the IL-3-mediated proliferation of cells expressinga receptor for IL-3, comprising the step of contacting said cells withan effective amount of an hIL-3 variant according to claim
 6. 17. Amethod to antagonize the IL-3-mediated differentiation of cellsexpressing a receptor for IL-3, comprising the step of contacting saidcells with an effective amount of an hIL-3 variant according to claim 6.18. A method to antagonize the IL-3-mediated functional activation ofcells expressing a receptor for IL-3, comprising the step of contactingsaid cells with an effective amount of an hIL-3 variant according toclaim
 6. 19. A method according to any one of claims 16, 17, and 18,wherein said cells are present in a patient, and the method comprisesthe step of administering said variant to said patient.